鸡补体受体2基因克隆、蛋白表达纯化及其多克隆抗体的制备和鉴定  

Cloning,Protein Expression and Purification of Chicken Complement Receptor 2 Gene and Preparation and Identification of Its Polyclonal Antibody

作　　者：孔子萌 江波 蔡云虹 何后军[1] 李永清[2] 靳换 KONG Zimeng;JIANG Bo;CAI Yunhong;HE Houjun;LI Yongqing;JIN Huan(College of Animal Science and Technology,Jiangxi Agricultural University,Nanchang 330045,China;Research Center for Infectious Diseases in Livestock and Poultry,Beijing Academy of Agriculture and Forestry Sciences,Beijing 100097,China)

机构地区：[1]江西农业大学动物科学技术学院,南昌330045 [2]北京市农林科学院,畜禽疫病研究中心,北京100097

出　　处：《中国畜牧兽医》2020年第1期201-208,共8页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金面上项目(31672588);北京市博士后科研活动经费资助项目(2018-XX-053);北京市农林科学院博士后基金(2018-ZZ-005);北京市农林科学院科技创新能力建设专项(KJCX201914)

摘　　要：为初步鉴定鸡补体受体2(ChCR2)基因,本试验设计特异性引物,利用RT-PCR方法扩增ChCR 2基因。利用同源臂连接的方法构建去掉跨膜区的ChCR2-△TM基因的原核表达载体pGEX-6P-1-ChCR2-△TM、pET-32a-ChCR2-△TM和真核表达载体pCMV-HA-ChCR2-△TM,将两个原核质粒转入大肠杆菌表达系统中表达GST-ChCR2-△TM和His-ChCR2-△TM蛋白。将纯化后的GST-ChCR2-△TM蛋白免疫BALB/c小鼠制备血清多克隆抗体,以His-ChCR2-△TM蛋白为包被原,ELISA检测血清多克隆抗体效价。利用间接免疫荧光试验和Western blotting对抗原抗体的特异结合性进行鉴定。结果显示,扩增到了一条大小为1113 bp的ChCR 2基因条带。成功构建了去掉跨膜区的原核表达载体pGEX-6P-1-ChCR2-△TM、pET-32a-ChCR2-△TM和真核表达载体pCMV-HA-ChCR2-△TM。诱导表达的GST-ChCR2-△TM蛋白在低温(16℃)诱导条件下可溶,而His-ChCR2-△TM蛋白以包涵体的形式存在。经ELISA测定,GST-ChCR2-△TM蛋白免疫的BALB/c小鼠血清效价为1∶512000。间接免疫荧光试验和Western blotting结果显示,制备的抗GST-ChCR2-△TM蛋白血清多克隆抗体可与ChCR2蛋白发生特异性结合。本研究结果为ChCR 2基因的进一步鉴定及鸡乃至禽类的相关免疫学研究提供了参考依据。In order to identify chicken complement receptor 2(ChCR2)gene,we designed specific primers and amplified ChCR 2 gene by RT-PCR.The prokaryotic expression vectors pGEX-6P-1-ChCR2-△TM and pET-32a-ChCR2-△TM and the eukaryotic expression vector pCMV-HA-ChCR2-△TM were constructed by homologous arm ligation.The two prokaryotic plasmids were transferred into E.coli expression system to express GST-ChCR2-△TM and His-ChCR2-△TM proteins.The polyclonal antibody was prepared by immunizing BALB/c mice with GST-ChCR2-△TM protein.The antibody titer was detected by ELISA.Indirect immunofluorescence test and Western blotting were used to identify the specific binding of antigen and antibody.The results showed that a ChCR 2 gene with a size of 1113 bp was amplified.The prokaryotic expression vectors pGEX-6P-1-ChCR2-ΔTM and pET-32a-ChCR2-ΔTM and the eukaryotic expression vector pCMV-HA-ChCR2-△TM with the transmembrane region removed were successfully constructed.The GST-ChCR2-△TM protein was soluble at low temperature(16℃),while His-ChCR2-△TM protein was in the form of inclusion body.The serum titer of BALB/c mice immunized with GST-ChCR2-△TM protein was 1∶512000 detected by ELISA.The results of indirect immunofluorescence assay and Western blotting showed that the polyclonal antibody against GST-ChCR2-△TM protein could specifically bind to ChCR2 protein.The results of this study provided a reference for further identification of ChCR 2 gene and related immunological research of chicken and even poultry.

关 键 词：ChCR2基因 蛋白表达和纯化 多克隆抗体 ELISA 间接免疫荧光 Western BLOTTING 

分 类 号：S831[农业科学—畜牧学]